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Chlamydophila pneumoniae induces expression of Toll-like Receptor 4 and release of TNF-α and MIP-2 via an NF-κB pathway in rat type II pneumocytes

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The role of alveolar type II cells in the regulation of innate and adaptive immunity is unclear. Toll-like receptors (TLRs) have been implicated in host defense. The purpose of the present study was to investigate whether Chlamydophila pneumoniae (I) alters the expression of TLR2 and/orTLR4 in type II cells in a (II) Rho-GTPase- and (III) NF-κB-dependent pathway, subsequently (IV) leading to the production of (IV) pro-inflammatory TNF-α and MIP-2. Methods Isolated rat type II pneumocytes were incubated with C. pneumoniae after pre-treatment with calcium chelator BAPTA-AM, inhibitors of NF-κB (parthenolide, SN50) or with a specific inhibitor of the Rho-GTPase (mevastatin). TLR2 and TLR4 mRNA expressions were analyzed by PCR. Activation of TLR4, Rac1, RhoA protein and NF-κB was determined by Western blotting and confocal laser scan microscopy (CLSM) and TNF-α and MIP-2 release by ELISA. Results Type II cells constitutively expressed TLR4 and TLR2 mRNA. A prominent induction of TLR4 but not TLR2 mRNA was detected after 2 hours of incubation with C. pneumoniae. The TLR4 protein expression reached a peak at 30 min, began to decrease within 1–2 hours and peaked again at 3 hours. Incubation of cells with heat-inactivated bacteria (56°C for 30 min) significantly reduced the TLR4 expression. Treated bacteria with polymyxin B (2 μg/ml) did not alter TLR4 expression. C. pneumoniae-induced NF-κB activity was blocked by TLR4 blocking antibodies. TLR4 mRNA and protein expression were inhibited in the presence of BAPTA-AM, SN50 or parthenolide. TNF-α and MIP-2 release was increased in type II cells in response to C. pneumoniae, whereas BAPTA-AM, SN50 or parthenolide decreased the C. pneumoniae-induced TNF-α and MIP-2 release. Mevastatin inhibited C. pneumoniae-mediated Rac1, RhoA and TLR4 expression. Conclusion The TLR4 protein expression in rat type II cells is likely to be mediated by a heat-sensitive C. pneumoniae protein that induces a fast Ca 2+ -mediated NF-κB activity, necessary for maintenance of TLR4 expression and TNF-α and MIP-2 release through possibly Rac and Rho protein-dependent mechanism. These results indicate that type II pneumocytes play an important role in the innate pulmonary immune system and in inflammatory response mechanism of the alveolus.

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Published 01 January 2005
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Language English
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Respiratory Research
BioMedCentral
Open Access Research Chlamydophila pneumoniae induces expression of Toll-like Receptor 4 and release of TNF-and MIP-2 via an NF-B pathway in rat type II pneumocytes 1 12 3 Heide Wissel*, Christian Schulz, Petra Koehne, Ekkehard Richter, 4 1,5 Matthias Maassand Mario Rüdiger
1 2 Address: Clinicfor Neonatology, Campus Charité Mitte, Schumannstr. 20–21, D10098 Berlin, Germany,Clinic for Neonatology, Campus 3 4 Charité VirchowKlinikum, Berlin, Germany,Department of Cell Biology, Institute of Biology, HumboldtUniversity Berlin, Germany,SALK 5 Microbiology, Salzburger Landeskliniken, MuellnerHauptstr. 56, A5020 Salzburg, Austria andClinic for Neonatology, Medical University Innsbruck, Austria Email: Heide Wissel*  hwissel@charite.de; Christian Schulz  lonyx@gmx.de; Petra Koehne  petra.koehne@charite.de; Ekkehard Richter  ekkehard.richter1@rz.huberlin.de; Matthias Maass  m.maass@salk.at; Mario Rüdiger  mario.ruediger@uibk.ac.at * Corresponding author
Published: 03 June 2005Received: 05 April 2005 Accepted: 03 June 2005 Respiratory Research2005,6:51 doi:10.1186/1465-9921-6-51 This article is available from: http://respiratory-research.com/content/6/1/51 © 2005 Wissel et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Chlamydophila (Chlamydia) pneumoniaerat type II pneumocytesTLR4NFBcytokines
Abstract Background:The role of alveolar type II cells in the regulation of innate and adaptive immunity is unclear. Toll-like receptors (TLRs) have been implicated in host defense. The purpose of the present study was to investigate whether Chlamydophila pneumoniae (I) alters the expression of TLR2 and/orTLR4 in type II cells in a (II) Rho-GTPase- and (III) NF-B-dependent pathway, subsequently (IV) leading to the production of (IV) pro-inflammatory TNF-and MIP-2. Methods:Isolated rat type II pneumocytes were incubated with C. pneumoniae after pre-treatment with calcium chelator BAPTA-AM, inhibitors of NF-B (parthenolide, SN50) or with a specific inhibitor of the Rho-GTPase (mevastatin). TLR2 and TLR4 mRNA expressions were analyzed by PCR. Activation of TLR4, Rac1, RhoA protein and NF-B was determined by Western blotting and confocal laser scan microscopy (CLSM) and TNF-and MIP-2 release by ELISA. Results:Type II cells constitutively expressed TLR4 and TLR2 mRNA. A prominent induction of TLR4 but not TLR2 mRNA was detected after 2 hours of incubation with C. pneumoniae. The TLR4 protein expression reached a peak at 30 min, began to decrease within 1–2 hours and peaked again at 3 hours. Incubation of cells with heat-inactivated bacteria (56°C for 30 min) significantly reduced the TLR4 expression. Treated bacteria with polymyxin B (2µg/ml) did not alter TLR4 expression. C. pneumoniae-induced NF-B activity was blocked by TLR4 blocking antibodies. TLR4 mRNA and protein expression were inhibited in the presence of BAPTA-AM, SN50 or parthenolide. TNF-MIP-2 release was increased in type II cells in and response to C. pneumoniae, whereas BAPTA-AM, SN50 or parthenolide decreased the C. pneumoniae-induced TNF-and MIP-2 release. Mevastatin inhibited C. pneumoniae-mediated Rac1, RhoA and TLR4 expression. Conclusion:The TLR4 protein expression in rat type II cells is likely to be mediated by a heat-sensitive C. pneumoniae protein 2+ that induces a fast Ca-mediated NF-B activity, necessary for maintenance of TLR4 expression and TNF-and MIP-2 release through possibly Rac and Rho protein-dependent mechanism. These results indicate that type II pneumocytes play an important role in the innate pulmonary immune system and in inflammatory response mechanism of the alveolus.
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